Luminal B型局部晚期乳腺癌新辅助化疗疗效预测基因的研究

目的 筛选Luminal B型局部晚期乳腺癌新辅助化疗疗效预测基因。方法 收集10例Luminal B型乳腺癌患者,均行DA方案新辅助化疗（多西他赛 75mg／m2静滴,d1;表阿霉素80mg／m2 静滴d1,21天为1周期,共4个周期）,根据化疗疗效分为完全病理缓解（pCR）组（n=3）和非完全病理缓解（npCR）组（n=7）。采用人基因表达谱cDNA芯片从10例乳腺癌患者中筛选新辅助化疗疗效相关基因,荧光定量PCR验证差异表达基因。结果 pCR组与npCR组癌组织相比,上调基因231个,下调基因195个;pCR组癌组织与癌旁组织相比,上调的基因357个,下调的基因544个;npCR组癌组织与癌旁组织相比,上调基因143个,下调基因101个;pCR组与npCR组癌旁组织比较,上调基因98个,下调基因67个。pCR组与npCR组癌组织比较,筛选出6个与肿瘤有关的基因,其中上调基因为CYP4Z1、FGFL6、BCAR4,下调基因为FABP4、S100B、ALPH1L1。荧光定量PCR进一步验证显示,两组mRNA表达差异的基因为CYP4Z1和BCAR4,pCR组两种基因表达均显著低于npCR组（P＜0.05）。结论 对局部晚期Luminal B型乳腺癌低表达BCAR4和CYP4Z1的患者新辅助治疗选择采用DA方案化疗更有可能获得pCR。     

p53 and P-glycoprotein are often co-expressed and are associated with poor prognosis in breast cancer.

Expression of both P-glycoprotein (P-gp) and mutant p53 have recently been reported to be associated with poor prognosis of breast cancer. The expression of P-gp is associated in vitro and in vivo with cross-resistance to several anti-cancer drugs. p53 plays a regulatory role in apoptosis, and mutant p53 has been suggested to be involved in drug resistance. Interestingly, in vitro experiments have shown that mutant p53 can activate the promoter of the MDR1 gene, which encodes P-gp. We investigated whether p53 and P-gp are simultaneously expressed in primary breast cancer cells and analysed the impact of the co-expression on patients prognosis. Immunohistochemistry was used to investigate P-gp expression (JSB-1, C219) and nuclear p53 accumulation (DO-7) in 20 operable chemotherapy untreated and 30 locally advanced breast cancers undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. Double immunostaining showed that P-gp expression and nuclear p53 accumulation often occur concomitantly in the same tumour cells. A correlation between p53 and P-gp expression was found in all 50 breast cancers (P = 0.003; Fisher''s exact test). P-gp expression, nuclear p53 accumulation, and co-expression of p53 and P-gp were more frequently observed in locally advanced breast cancers than in operable breast cancers (P = 0.0004, P = 0.048; P = 0.002 respectively. Fisher''s exact test). Co-expression of p53 and P-gp was the strongest prognostic factor for shorter survival by multivariate analysis (P = 0.004) in the group of locally advanced breast cancers (univariate analysis: P = 0.0007). Only 3 out of 13 samples sequentially taken before and after chemotherapy displayed a change in P-gp or p53 staining. In conclusion, nuclear p53 accumulation is often associated with P-gp expression in primary breast cancer, and simultaneous expression of p53 and P-gp is associated with shorter survival in locally advanced breast cancer patients. Co-expression of P-gp and mutant p53 belong to a series of molecular events resulting in a more aggressive phenotype, drug resistance and poor prognosis.     

Clinical significance of the relationship between expression of survivin and effects of neoadjuvant chemotherapy in locally advanced breast cancer

OBJECTIVE: Explore the relationship between the expression intensity of survivin and the effectiveness of neoadjuvant chemotherapy in locally advanced breast cancer patients. METHODS: Neoadjuvant chemotherapy with epirubicin plus paclitaxel was administered to 76 patients in locally advanced breast cancer (including 25 cases of stage IIa, 26 of stage IIb, 16 of stage IIIa, and 9 of stage IIIb), the mean age is 52.8(33-79)years old. All patients were female. They were treated with epirubicin 60 mg/m(2), on day 1, by i. v. followed paclitaxel 175 mg/m(2) by 3 hours continues infusion on day 2 and every 3 weeks repeatedly. Premedication of dexamethasone, ondansetron, diphenhydramine and cimetidine were administered to prevent gastroenteric and allergic reactions before chemotherapy. Four cycles were used. The expression of survivin in breast cancer tissue was detected with SDS-PAGE, western-immunoblotting and immunohistochemistry (IHC), and then that were immunological stained by anti survivin monoclonal antibody, and also the results were analyzed for the relationship between the expressed intensity of survivin and the effect of neoadjuvant chemotherapy in locally advanced breast cancer patients. RESULTS: Nineteen out of 76 patients had a clinical complete response, 36 had clinical partial response, and 21 had no change. The response rate was 72.37%(55/76). We found survivin could be differently expressed in 76 patients with SDS-PAGE, western-immunoblotting and IHC and then immune stain by anti survivin monoclonal antibody. Forty six patients were low expressed of survivin and 9 patients were high expressed in all response patients. Eight patients were low expressed, only 1 patient was high expressed of survivin in 9 patients had pCR. But no finding the relationship between the expression of survivin and TNM stage, ER, PgR, HER-2. CONCLUSION: The patients have high response rate of low expression of survivin after neoadjuvant chemotherapy with TE regimen in locally advanced breast cancer patients. This results shows that survivin is an important predictive factor for effectiveness of neoadjuvant chemotherapy with TE regimen in locally advanced breast cancer.     

Changes in gene expression associated with response to neoadjuvant chemotherapy in breast cancer.

PURPOSE: At present, clinically useful markers predicting response of primary breast carcinomas to either doxorubicin-cyclophosphamide (AC) or doxorubicin-docetaxel (AD) are lacking. We investigated whether gene expression profiles of the primary tumor could be used to predict treatment response to either of those chemotherapy regimens. PATIENTS AND METHODS: Within a single-institution, randomized, phase II trial, patients with locally advanced breast cancer received six courses of either AC (n = 24) or AD (n = 24) neoadjuvant chemotherapy. Gene expression profiles were generated from core-needle biopsies obtained before treatment and correlated with the response of the primary tumor to the chemotherapy administered. Additionally, pretreatment gene expression profiles were compared with those in tumors remaining after chemotherapy. RESULTS: Ten (20%) of 48 patients showed a (near) pathologic complete remission of the primary tumor after treatment. No gene expression pattern correlating with response could be identified for all patients or for the AC or AD groups separately. The comparison of the pretreatment biopsy and the tumor excised after chemotherapy revealed differences in gene expression in tumors that showed a partial remission but not in tumors that did not respond to chemotherapy. CONCLUSION: No gene expression profile predicting the response of primary breast carcinomas to AC- or AD-based neoadjuvant chemotherapy could be detected in this interim analysis. More subtle differences in gene expression are likely to be present but can only be reliably identified by studying a larger group of patients. Response of a breast tumor to neoadjuvant chemotherapy results in alterations in gene expression.     

基于单细胞测序分析肿瘤相关巨噬细胞来源的半乳糖凝集素3的功能

目的：从肿瘤相关巨噬细胞（TAM）在化疗前后的表型变化入手,寻找通过调控肿瘤免疫微环境（TIME）从而影响肿瘤治疗效果和预后的功能分子。方法：利用欧洲核苷酸数据库（ENA）的PRJEB45598数据集,分析进展期胃癌患者化疗前后活检肿瘤组织单细胞测序数据,采用主成分分析（PCA）和一致流形近似与投影（UMAP）降维,获得31个亚群细胞,并进一步进行TAM亚型分析、差异基因筛选,寻找化疗后M2型TAM中高表达的基因。通过黑色素瘤B16-F10细胞皮下移植瘤模型验证化疗前后特定基因m RNA和蛋白水平表达变化,并通过Incucyte体外分析该蛋白是否调控化疗药物诱导的肿瘤细胞死亡。结果：聚焦单细胞测序数据中M2型TAM的特征表达基因,发现半乳糖凝集素3(LGALS3)在胃癌化疗后m RNA水平显著升高（P<0.01）,在多种肿瘤中LGALS3高表达且与患者生存期呈负相关（P<0.05或P<0.01）。黑色素瘤B16-F10细胞移植瘤模型中,LGALS3在M2型TAM中高表达（P<0.01）,且奥沙利铂化疗后表达进一步升高（P<0.05）。体外对肿瘤细胞给予重组...     

Expression of drug resistance proteins in breast cancer,in relation to chemotherapy

Drug resistance plays an important role in chemotherapy failure in breast cancer. We studied the expression of MDR1, MRP, LRP, DNA topoisomerases, p53 and Ki-67 in different groups of breast cancer patients in relation to chemotherapy. Tissues from 6 normal breasts and 20 primary operable, 40 locally advanced and 10 anthracycline-resistant metastatic breast cancers were assessed. Sequential samples of the same patient were available from 17 patients with locally advanced breast cancer undergoing neo-adjuvant chemotherapy and in 7 metastatic patients undergoing paclitaxel treatment. Protein expression was investigated by immunohistochemistry. Significantly higher protein expression was observed for Pgp, Ki-67 and p53 in the locally advanced breast cancers than in primary operable breast cancers. No other significant differences in protein expression were found among the 3 breast cancer groups. Expression of none of the markers that could be assessed (Pgp, MRP, LRP, p53 and Ki-67) in locally advanced breast cancer had predictive value for pathological response. Interestingly, after chemotherapy a significant decrease in percentage of Ki-67 positive tumor cells was observed, whereas the other markers did not vary substantially. Furthermore, considering all breast cancer samples, a cumulative dose of doxorubicin >400 mg/m² inversely correlated with Ki-67 positivity. However, 2 patients with a pathological complete remission had only 5-10% Ki67-positive tumor cells before chemotherapy, indicating that Ki67 negativity itself is not responsible for chemoresistance. In conclusion, none of the known proteins related to multidrug resistance predicted response to chemotherapy in breast cancer, and resistant clones left behind generally had a low proliferation rate.Int. J. Cancer 71: 787-795, 1997. © 1997 Wiley-Liss Inc.     

局部进展期乳腺癌新辅助化疗前后生物标志物表达变化与疗效的相关性

目的：探讨局部进展期乳腺癌行新辅助化疗前后相关生物标志物的表达变化情况与化疗疗效的相关性。方法：采用免疫组化方法检测102例新辅助化疗前后局部进展期乳腺癌组织中雌激素受体（ER）、孕激素受体（PR）、人类表皮生长因子受体－2（HER －2）、p53和增殖细胞核抗原（Ki －67）等表达,分析化疗前后生物标志物表达变化与化疗疗效的相关性。结果：ER 阴性组、PR 阴性组、Ki －67高表达组的新辅助化疗有效率分别为50．0％、49．1％、51．4％,高于 ER 阳性组26．0％、PR 阳性组25．5％、Ki －67低表达组9．4％（P ＜0．05）。Logistic 多因素回归分析显示,ER、Ki －67的表达水平是评估化疗疗效的独立因素（P ＜0．05）。Luminal 型乳腺癌总生存期高于 non －Luminal 型（Long －rank 检验,P ＜0．05）。结论：ER、Ki －67、分子亚型可作为局部进展期乳腺癌新辅助化疗疗效判断的重要预测指标。     

Role of ABCB1 and ABCC1 Gene Induction on Survival in Locally Advanced Breast Cancer

Abstract

Drug resistance to chemotherapy in patients with locally advanced breast cancer results in a decrease in treatment efficacy and in patient survival. This study aimed to evaluate the impact of ABCB1 and ABCC1 gene induction during chemotherapy on disease-free and overall survival of breast cancer patients.

Patients with locally advanced breast cancer were prospectively included. All patients were preoperatively treated with chemotherapy and underwent mastectomy. ABCB1 and ABCC1 gene and protein expressions were evaluated both before and after chemotherapy and investigated as molecular predictive parameters affecting disease free and overall survival. ABCB1 and ABCC1 gene expressions were evaluated with RTPCR following RNA isolation from tissue samples. P-glycoprotein and MRP1 in tissues were detected using immunohistochemistry.

Twenty-five female patients treated with either doxorubicin or epirubicin were included. Median follow-up time was 36 months during which 11 patients (44%) had recurrence, all of whom died. Mean disease-free survival for patients with and without ABCB1 gene induction was 13 and 55 months (p=0.0004), respectively, whereas overall survival was 21 and 57 months (p=0.0025), respectively. Mean disease-free survival for patients with and without ABCC1 gene induction was 32 and 48 months (p=0.97), respectively, and overall survival was 43 and 49 months (p=0.36), respectively.

ABCB1 gene induction decreases disease-free and overall survival in patients with locally advanced breast cancer due to anthracycline resistance. Detecting ABCB1 gene expression during chemotherapy helps to increase the efficacy of drug treatment by choosing the appropriate drugs resulting in prolonged survival.     

Intra-arterial infusion chemotherapy as an induction therapy in multidisciplinary treatment for locally advanced breast cancer. A long-term follow-up study

A multidisciplinary treatment including intra-arterial infusion chemotherapy as an induction therapy was administered to 55 patients with locally advanced breast cancer. Intra-arterial chemotherapy conducted preoperatively produced marked responses in primary and lymph node lesions with 78% complete + partial response (CR + PR), subsequently permitting extended radical mastectomy. Histologic examination of resected specimens also revealed that 33% of the patients had no viable cancer cells remaining in their lesions. Five-year and 10-year survival rates were 57% and 41%, respectively, compared with 24% and 18%, respectively for the 17 patients of historic control. Patients showing better local responses to intra-arterial chemotherapy had longer survival time with less frequent local recurrences. Intra-arterial chemotherapy is an effective modality for the treatment of locally advanced breast cancer.     

miR-27a在三阴性乳腺癌中的表达及其对细胞耐药的影响

目的:探讨miR-27a在三阴性乳腺癌（triple-negative breast cancer,TNBC）中的表达及其对细胞耐药的影响。方法:首先通过QRT-PCR检测TNBC细胞株及非TNBC细胞株中miR-27a及P-gp的差异表达;上调TNBC细胞中的miR-27a的表达,通过CCK8检测细胞对化疗药物敏感性的变化。同时收集TNBC患者化疗前后血液标本,将其分为化疗敏感组和化疗耐药组,QRT-PCR检测患者血液标本中miR-27a及P-gp的表达,分析miR-27a与乳腺癌患者预后相关性。结果:miR-27a在TNBC细胞株中的表达明显低于非TNBC细胞株,上调TNBC细胞株中miR-27a的表达能够降低P-gp的表达,增加细胞对化疗药物的敏感性,此外TNBC组中miR-27a的表达与患者组织学分级、临床分期及淋巴结转移相关（P<0.05）;在非TNBC组中miR-27a的表达与患者临床病理特征无明显相关性（P均＞0.05）。结论:miR-27a参与调节TNBC细胞耐药,miR-27a可作为评估乳腺癌化疗敏感性及临床预后的潜在靶基因。     

Preoperative chemotherapy for advanced breast cancer

Preoperative chemotherapy was initiated for breast cancer in an effort to decrease the number of viable cancer cells that were released into the blood stream during surgical procedure. This possibility was substantiated by several observations made in animal experiments and clinical studies. Preoperative chemotherapy was also given to render the advanced disease amenable to surgical intervention. In one report, systemic chemotherapy (CAF) for advanced breast cancer produced a response rate of 86% preoperatively, facilitating subsequent mastectomy and a postoperative 5-year survival rate of 52%. However no definite conclusion has yet been obtained as to the prognostic significance of systemic chemotherapy give preoperatively, and further comparative studies are therefore required. Preoperative intra-arterial chemotherapy as an induction therapy was administered to patients with locally advanced breast cancer including inflammatory breast cancer, the treatment developed in Japan. In our institute, intra-arterial chemotherapy with ADR or MMC plus 5-FU resulted in a marked decrease in the size of primary and lymph node lesions with 82% CR + PR. Histological examination of resected specimens also revealed that 35% of the patients had no viable cancer cells remaining in their lesions. Five-year and 10-year survival rates were 57% and 41%, respectively, compared with 24% and 18%, respectively for historical controls. Patients showing better local responses to intra-arterial chemotherapy had longer survival time with less frequent local recurrences. Some other studies also indicated improved survival in locally advanced breast cancer as a result of preoperative intra-arterial chemotherapy. Preoperative chemotherapy including systemic administration is a promising modality for advanced breast cancer.